(M-484) Pulmonary Delivery of Levofloxacin Incorporated Extracellular Matrix Nanoparticles for Cystic Fibrosis Infections

Introduction:: Cystic fibrosis (CF) is a progressive genetic disorder that affects around 40,000 people per year. This disease results from a mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) protein. The mutation causes dysregulated transport of sodium and chloride ion movement across cell membranes, causing the mucus in the lungs and many other organs, to become more viscous. Thick and sticky mucus in the lung makes it easier for pathogens to invade and cause persistent infections and chronic inflammation that leads to airway remodeling. Pseudomonas aeruginosa and Staphylococcus aureus are two of the most common infections in CF patients that are often resistant to antibiotics. One antibiotic approved to treat these infections is Levofloxacin (LVX) which functions to inhibit bacterial replication. However due to the increased antibiotic resistance of current therapies, new treatments have employed nanoparticle formulations for enhanced targeting and delivery to these pathogens. Previously, porcine lung decellularized extracellular matrix (dECM) nanoparticles (NPs) have demonstrated pro-regenerative, non-toxic, and antimicrobial effects in vitro, capable of targeting the lower respiratory tract. Therefore, we hypothesize that a combination of LVX and ECM formed into nanoparticles may be able to penetrate through CF mucus to inhibit bacterial growth and may induce pro-regenerative responses for airway remodeling. Treating cystic fibrosis infections will ultimately decrease patient costs and hospital visits while also increasing their quality of life.

Materials and Methods:: Briefly, LVX and porcine lung decellularized ECM powders were dissolved, drawn up into a syringe and attached to a blunt tip needle. The nanoparticles were electrosprayed using a syringe pump attached to a high voltage generator with set parameters for flow rate, applied voltage, and height. Ratios of 1:1 LVX to ECM and a 10:1 LVX to ECM were sprayed using the same parameters. The nanoparticles were then characterized for size and charge using scanning electron microscopy (SEM) and a ZetaSizer. Release kinetics of LVX were analyzed using in vitro dissolution that mimics the native lung, followed by high performance liquid chromatography (HPLC) assays. Bacterial reduction assays were performed using P. aeruginosa and E.Coli bacterial lines with different concentrations of the NPs compared to LVX alone.

Results, Conclusions, and Discussions:: We found that the nanoparticles morphology from SEM imaging (Figure 1) represented mostly spherical-like particles with some crystal structures most likely formed from the LVX antibiotic. The images also showed a range in sizes which may mean that some smaller particles will not only penetrate through the mucus but may also deposit in the lower respiratory tract, possibly preventing further bacterial infection or alveolar remodeling from inflammation. From both SEM imaging and the ZetaSizer, our particles were shown to have an average diameter of 223 nm. If particles are less than 300 nm they should be able to penetrate through the mucus to target the bacteria as CF mucus pore sizes are much smaller than healthy lungs ( > 400 nm).The nanoparticles had an average zeta-potential of -3.05 mV, which falls within the neutral range, shown in Figure 2. The neutral nature of the particles may result in the particles being taken up by the CF mucus to penetrate to the bacteria as highly positive or highly negative particles are likely to repel CF sputum. HPLC revealed that our NPs have an almost immediate release and continues to release slowly over time. Surprisingly, this immediate release may be able to penetrate through the mucus much easier than slow releasing particles. The HPLC assay also showed consistency within our formulated product batches. Through our bacterial reduction assays (Figure 3), our results showed that NPs plated with a higher concentration of 1:1 LVX-ECM NPs show greater bacterial inhibition than both high and low concentrations of 10:1 LVX-ECM NPs. This further shows the antibacterial properties of the ECM, especially when combined with LVX.

Overall, these nanoparticles show promise for a potential CF therapeutic that is able to penetrate through mucus to target pathogens like P. aeruginosa. The validation of the nanoparticles characteristics, size and zeta potential, may set up further studies through CF mucus assays that will further confirm their penetrating abilities. We may also look to investigate Staphylococcus aureus, a common infection among younger CF patients. Further studies may also include analyzing the aerodynamic diameter of the nanoparticles for further deposition characterization.

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